The field of total internal reflection microscopy makes use of the refraction behavior of light during the transition from an optically denser medium to an optically thinner medium. Thus, for instance, the transition from cover glass (n1=1.518) to wear (n2=1.33) yields a critical angle of 61°, the angle of total reflection. Under the conditions of total reflection (angle≧61°), a stationary evanescent wave forms in the medium having the lower refractive index. The intensity of this wave decreases exponentially with the distance from the boundary surface. For this reason, fluorophores situated further away from the boundary surface are not excited. The background fluorescence is drastically reduced. The image contrast is improved in this process while, at the same time, the resolution is considerably increased. A prerequisite for utilizing the above-mentioned phenomenon is a sufficiently large difference in the refractive indices of the cover glass and the medium.
U.S. patent application publication no. 2002/0097489 discloses a microscope involving the evanescent illumination of a specimen. The microscope comprises a white-light source whose light is coupled into the specimen side via a slit diaphragm through the microscope objective for purposes of evanescent illumination. The illumination light propagets itself in the specimen slide as a result of total internal reflection, a process in which the specimen is only illuminated in the area of the evanscent field that projects from the specimen side. Microscopes of this type are known under the designation TIRFM (Total Internal Reflection Fluorescent Microscope).
The z-resolution of TIRFMs is outstanding due to the fact that the evanescent field projects only about 100 nm into the specimen.
German patent application DE 101 08 796 A1 discloses a high-aperture objective, especially for TIRF applications. The objective consists of a first lens having positive refractive power, a second lens having negative refractive power, whereby the focal length ratio between the two lenses lies within the range from −0.4 to −0.1 and the total refractive power is greater than zero. Moreover, the objective comprises two positive lenses whose diameter-to-focal length ratio is greater than 0.3 and smaller than 0.6. Furthermore, the objective comprises a negative lens and a collective lens, whereby the negative lens faces the front group and the focal length ratio of the negative lens to the collective lens iles between −0.5 and −2.
German patent application DE 102 17 098 A1 discloses an incident-illumination arrangement for TIRF microscopy. This incident-illumination arrangement comprises a source of illumination that, during operation, emits a polarized illuminating bundle of rays that propagates itself at an angle relative to the optical axis, and a deflection device that deflects the illuminating bundle of rays and couples it into the objective parallel to the optical axis. With this incident-illumination arrangement, it is provided that the illuminating bundle of rays emitted by the source of illumination has s-polarization and p-polarization directions having the phase differential and the deflection device reflects the illuminating bundle of rays x times, wherein x=(n=180°−d)/60°.
German pat. appl. DE 101 43 481 A1 discloses a microscope for TIRM Total Internal Reflection Microscopy). The microscope has a housing and an objective. The illumination light emitted by an illumination device can be coupled in via an adapter that can be slid into the microscope housing.
U.S. patent application publication no. 2004/0001253 discloses a microscope with an optical illumination system that allows a simple switchover between evanescent illumination and reflection illumination. The illumination system comprises a laser light source whose light is coupled into an optical fiber. An outcoupling optical system is also provided that focuses the light emerging from the fiber into a rear focal point of the microscope objective. The optical fiber can be moved in a plane perpendicular to the optical axis of the microscope objective.
German patent application DE 102 29 935 A1 discloses a device for coupling light into a microscope. There, laser light is directed at the preparation in the plane of the illuminated field diaphragm through a light-conductive fiber coupler configured as a slide. The invention is particularly well-suited for the TIRF method.
In scanning microscopy, a specimen is illuminated with a light beam so that the detection light emitted by the specimen can be observed as reflection or fluorescent light. The focus of an illuminating bundle of rays is moved in a plane of the specimen by means of a controllable beam deflector, generally by tilting two mirrors, whereby the deflection axes are usually positioned perpendicular to each other, so that one mirror deflects in the x direction while the other deflects in the y-direction. The mirrors are tilted, for example, employing galvanometric actuating elements. The power of the detection light coming from the object is measured as a function of the position of the scanning beam. Normally, the actuating elements are equipped with sensors to ascertain the actual position of the mirror. Especially in confocal scanning microscopy, an object is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light from the source is focused into a pinhole diaphragm—the so-called excitation diaphragm—a beam splitter, a beam deflector to control the beam, a microscope optical system, a detection diaphragm and the detectors for picking up the detection or fluorescent light. The illumination light is coupled in via a beam splitter. Via the beam deflector, the fluorescent or reflection light coming from the object returns to the beam splitter, passes through it and is subsequently focused onto the detection diaphragm behind which the detectors are located. This detector arrangement is called a descan arrangement. Detection light that does not stem directly from the focus region takes a different light path and does not pass the detection diaphragm, so that point information is obtained that yields a three-dimensional image as a result of the sequential scanning of the object with the focus of the illuminating bundle of rays. For the most part, a three-dimensional image is attained by means of layer-by-layer image data acquisition.
The system known from the state of the art have the drawback that, in order to couple in the TIRF illumination light, they at times require optical systems in the beam path of the microscope that are very complex and that occupy a lot of space. This has a detrimental impact especially on the detection beam path and often causes a loss of detection light power.
As mentioned above, these are microscopic TIRF examinations, where a specimen is illuminated at a very flat angle, namely, in order to generate total reflection on the surface of the specimen. For this purpose, the state of the art employs objectives with a very large aperture in order to attain large illumination apertures. The fluorescence excitation is done with a laser light source whereby the laser is regularly imaged onto a point at the pupil rim of the objective. It is likewise known from the state of the art to use gas-discharge lamps for the fluorescence excitation. Here, an annular diaphragm is placed into the aperture plane of the illumination beam path, so that only the pupil rim of the objective is illuminated.
The technology implemented up until now for TIRF microscopy entails the disadvantage that it requires extremely precise adjustment of the deflection mirrors, diaphragms and the like. Extremely well-corrected illumination optical systems are needed in order to achromatically image the aperture diaphragm in the objective pupil. Thus, very special microscopes with an external fluorescent illumination and a laser arrangement have been employed so far.